Protein C-mannosylation is enzyme-catalysed and uses dolichyl-phosphate-mannose as a precursor.
C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is a novel kind of protein glycosylation that differs fundamentally from N- and O-glycosylation in the protein-sugar linkage. Previously, we established that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence -x-x-W, where the first Trp becomes C-mannosylated. Here we investigated the reaction with respect to the mannosyl donor and the involvement of a glycosyltransferase. C-mannosylation of Trp-7 was reduced 10-fold in CHO (Chinese hamster ovary) Lec15 cells, which are deficient in dolichyl-phosphate-mannose (Dol-P-Man) synthase activity, compared with wild-type cells. This was not a result of a decrease in C-mannosyltransferase activity. Rat liver microsomes were used to C-mannosylate the N-terminal dodecapeptide from RNase 2 in vitro, with Dol-P-Man as the donor. This microsomal transferase activity was destroyed by heat and protease treatment, and displayed the same acceptor substrate specificity as the in vivo reaction studied previously. The C-C linkage between the indole and the mannosyl moiety was demonstrated by tandem electrospray mass spectrometry analysis of the product. GDP-Man, in the presence of Dol-P, functioned as a precursor in vitro with membranes from wild-type but not CHO Lec15 cells. In contrast, with Dol-P-Man both membrane preparations were equally active. It is concluded that a microsomal transferase catalyses C-mannosylation of Trp-7, and that the minimal biosynthetic pathway can be defined as: Man -> -> GDP-Man -> Dol-P-Man -> (C2-Man-)Trp.
Doucey
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MA
Friedrich Miescher-Institut, CH-4002 Basel, Switzerland.
Hess
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Cacan
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Hofsteenge
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D000818:Animals; D016466:CHO Cells; D002244:Carbon; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D004287:Dolichol Monophosphate Mannose / Q000378:metabolism*; D004722:Endoribonucleases / Q000096:biosynthesis*; D006031:Glycosylation; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D006801:Humans; D008358:Mannose / Q000378:metabolism*; D008364:Mannosyltransferases / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D008956:Models, Chemical; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism
D010446:Peptide Fragments; D006155:Guanosine Diphosphate Mannose; D004287:Dolichol Monophosphate Mannose; D002244:Carbon; D014364:Tryptophan; D008364:Mannosyltransferases; C082696:protein O-mannosyltransferase; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D004722:Endoribonucleases; C029494:ribonuclease U; D008358:Mannose